Enveloped miroorganism

ABSTRACT

The invention relates to an enveloped microorganism in whose genome the following components are inserted and can be expressed: I) a nucleotide sequence that encodes a directly or indirectly, antiproliferatively or cytotoxically active expression product or a plurality of said expression products, II) a nucleotide sequence that encodes or is constitutively active for a blood plasma protein under the control of a activation sequence that can be activated in the microorganism, III) optionally, a nucleotide sequence that encodes or is constitutively active for a cell-specific ligand under the control of an activation sequence that can be activated in the microorganism, IV) a nucleotide sequence for a transport system that induces expression of the expression products of components I) and II) and optionally III) on the outer surface of the microorganism or that induces secretion of the expression products of component I) and expression of component II) and optionally component III) and that is preferably constitutively active, V) optionally a nucleotide sequence for a protein used for lysis of the microorganism in the cytosol of mammalian cells and for the intracellular release of plasmids with at least one or more components I) and VI) contained in the lysed microorganism, and VI) an activation sequence that can be activated in the microorganism, and/or that is tissue-specific, tumor cell-specific, function-specific or not cell-specific, for expressing component I). The inventive microorganism is further characterized in that any of components I) to VI) can be present either single or several times, and can be either identical or different.

FIELD OF THE INVENTION

The invention relates to a microorganism with foreign nucleotidesequences, by means of which antiproliferatively or cytotoxically actingexpression products can be expressed, and to the use of suchmicroorganisms for the production of pharmaceutical compositions, to aplasmid and a method for the production of such a microorganism, and tothe uses of such microorganisms.

BACKGROUND OF THE INVENTION AND PRIOR ART

Virulence-reduced microorganisms, such as genetically modified viruses,or virulence-attenuated bacteria gain increasing importance as carriersof foreign nucleic acid sequences to be used in the gene therapy.

For the gene therapy, the foreign nucleic acids are either inserted invitro into tissue cells, and these cells are administered to thepatient, or the microorganisms are injected to the patient, expectingthat the microorganisms transfer as gene ferries the foreign nucleicacid into the desired tissue cell.

Microorganisms are particles. After injection into an organism, theseparticles are mainly received by the so-called reticuloendothelialsystem. In order to achieve against this elimination mechanismnevertheless an enrichment of the microorganisms used as gene ferries ina target tissue, the microorganisms are provided with cell-specificligands. Up to now, in spite of this provision, the elimination of themicroorganisms by the reticuloendothelial system could only slightly bereduced.

An essential research aim of the gene therapy is the therapy ofproliferative diseases—such as tumors, leukemias, chronic inflammations,autoimmune diseases and rejections of transplanted organs, the treatmentof which is still insufficient, in spite of all successes of themedicament therapy. For instance, in spite of all successes of surgery,radiotherapy, chemotherapy and also immune therapy for the treatment oftumors, there could not be achieved up to now a healing of advancedtumors of the head and the neck, the central nervous system, the mammarygland, the lung, the gastrointestinal tract, the liver, the pancreas,the kidney, the skin, the ovaries and the prostate.

The reasons for this poor success of the tumor therapy are manifold andnot yet comprehensively known. To the main reasons, however, belong, i)before (primarily) existing resistances of the tumor cells against thein vivo achievable concentrations of chemotherapeutic agents, ofirradiations or against immunotherapeutic agents; ii) resistancesagainst the respective therapeutic agent generated in response to thetherapy. These induced so-called secondary resistances are caused by thegenetic variability of the tumor cells permitting them to avoid theeffects of the tumor therapeutic agents by the development of resistancemechanisms; iii) pharmacokinetic and/or pharmacodynamic insufficienciesof the up to now available tumor therapeutic agents, due to which theconcentration of the respective tumor therapeutic agent, irrespective ofwhether there are primary tumors, recidivations or metastases, isabsolutely too small to eliminate the tumor. To these insufficiencies ofthe tumor therapeutic agents belong, iv) a too high distribution volume;v) the insufficient enrichment at the tumor or at the tumor cells; vi)the insufficient penetration capability in the tumor; and/or vii) thetoxic effect on the total organism, which limits an increase of the dosefor an increased enrichment at the tumor.

In the past, different methods were used for trying to enrich tumortherapeutic agents at the tumor.

Tumor cell-specific ligands, for instance antibodies or the fissionproducts thereof, coupled to cytostatics, to antitumorally actingcytokines, to cytotoxic proteins, or to isotopes, did lead to anenrichment of the cytotoxic active substances at the tumor, compared tothe normal tissue, however this enrichment was in the by far most casesnot sufficient for a therapy of the tumor (survey: Sedlacek et al.,Contributions to Oncology 43:1-145, 1992; Carter, Nature Reviews Cancer1:118-129, 2001).

As a consequence, amplification systems have been designed, by means ofwhich the concentration of the respective active substance at the tumorcould be increased.

An amplification sequence had the aim to introduce such enzymes in thetumor, which were not generally accessible or foreign in the remainingbody, and which in turn could convert or split in the tumor a non-toxicpre-stage of a cytostatic into the cytotoxically active cytostatic. Theintroduction of the enzymes into the tumor was performed either byadministration of tumor cell-specific ligands, coupled to these enzymes(for instance in the form of the antibody derived enzyme-mediatedprodrug therapy; ADEPT), or by the administration of genes for theseenzymes by means of tumor cell-specific or not specific vectors (genederived enzyme-mediated prodrug therapy; GDEPT) (Sedlacek et al.,Contributions to Oncology 43:1-145, 1992; Sedlacek, Critical Reviews inOncology/Hematology 37:169-215, 2001; McCormick, Nature Reviews Cancer1:130-141, 2001; Carter, Nature Reviews Cancer 1:18-129, 2001).

The prior clinical investigations with ADEPT or GDEPT have furnishedinsufficient therapeutic results, however. As essential problems couldbe identified, i) the immunogenicity of a foreign enzyme; ii) therelatively small tumor localization rate of an antibody-enzyme conjugate(ADEPT); iii) the technical difficulties to produce fusion proteins froma humanized antibody with a human enzyme in a sufficiently large amountat acceptable costs; iv) the lacking tumor penetration of theantibody-enzyme conjugates or the gene vectors; and v) the too smalltransduction rate in vivo, i.e. the number of tumor cells of a tumornode, into which the genes for the enzyme could be expressed, was toosmall for a tumor-therapeutic effectivity.

Another amplification system is based on the induction of an immunereaction against tumor cells, in the course of which specificantibody-forming cells and cytotoxic cells proliferate. For theinduction of an immune reaction, tumor antigens are administered in asuitable preparation. It is the aim to break the immune toleranceagainst the tumor, this immune tolerance obviously existing in tumorpatients, and/or the resistance of the tumor against the own immunereaction.

Within the last decades, numerous technical variations of the tumorvaccination were clinically investigated by combination of differenttumor antigens with adjuvants, however without achieving the desiredbreak-through in the tumor therapy. New approaches, such as theadministration of combinations of immunogenic tumor-specific antigenswith new adjuvants, or of dendritic cells, loaded with tumor-specificantigens, or of nucleotide sequences that encode tumor-specificantigens, have resulted in first promising clinical results, however upto now there cannot be seen a break-through in the tumor therapy here,too.

A technique has been developed to express expression products of nucleicacid sequences introduced into bacteria on the cell membrane of thesebacteria or to have them secreted by these bacteria. The basis of thistechnique is the Escherichia coli hemolysin system hlyAs, whichrepresents the prototype of a type I secretion system of gram-negativebacteria. By means of the hlyAs, secretion vectors were developed, whichpermit an efficient discharge of protein antigens in Salmonellaenterica, Yersinia enterocolitica and Vibrio cholerae. Such secretionvectors contain the cDNA of an arbitrary protein antigen coupled to thenucleotide sequence for the hlyA signal peptide, for the hemolysinsecretion apparatus, hlyB and hlyD and the hly-specific promoter. Bymeans of this secretion vector, a protein can be expressed on thesurface of this bacterium. Thus genetically modified bacteria induce asvaccines a considerably higher immune protection than bacteria, in whichthe protein expressed by the introduced nucleic acid remainsintracellularly (Donner et al., EP 1015023 A, Gentschev et al., Gene179:133-140, 1996; Vaccine 19:2621-2618, 2001; Hess et al., PNAS93:1458-1463, 1996). The drawback of this method is however that byusing the hly-specific promoter the amount of the protein expressed onthe outer surface of the bacterium is extremely small.

A technique for the introduction of plasmid DNA into mammalian cells bycarrier bacteria such as Salmonella and Listeria monocytogenes has beendeveloped. Genes contained in these plasmids could be expressed in themammalian cells, even when they were under the control of a eukaryonticpromoter. Plasmids were introduced into Listeria monocytogenes germs,said plasmids containing a nucleotide sequence for an arbitrary antigenunder the control of an arbitrary eukaryontic promoter. By introductionof the nucleotide sequences for a specific lysis gene, it was achievedthat the Listeria monocytogenes germs dissolve in the cytosol of theantigen-presenting cell and release their plasmids, which then leads toexpression, processing and presentation of the plasmid-coded proteinsand clearly increases the immunogenicity of these proteins (Dietrich etal., Nat. Biotechnol. 16:181-185, 1998; Vaccine 19:2506-2512, 2001).

Virulence-attenuated variants of bacteria settling intracellularly havebeen developed. For instance such variants of Listeria monocytogenes,Salmonella enterica sv. typhimurium and typhi and BCG were already usedas well tolerated live vaccines against typhus and tuberculosis. Thesebacteria including their attenuated mutants are generally immunestimulating and can trigger a fair cellular immune response. Forinstance L. monocytogenes stimulates to a special degree via theactivation of TH1 cells the proliferation of cytotoxic lymphocytes.These bacteria supply secerned antigens directly into the cytosol ofantigen-presenting cells (APC; macrophages and dendritic cells), whichin turn express the costimulating molecules and trigger an efficientstimulation of T cells. The listeriae were in part degraded inphagosomal compartments, and the antigens produced by these carrierbacteria can therefore on the one hand be presented via MHC class IImolecules and thus lead to the induction of T helper cells. On the otherhand, the listeriae replicate after release from the phagosome in thecytosol of APCs; antigens produced and secerned by these bacteria aretherefore preferably presented via the MHC class I pathway, thereby CTLresponses against these antigens being induced. Furthermore, it could beshown that by the interaction of the listeriae with macrophages, naturalkiller cells (NK) and neutrophilic granulocytes, the expression of suchcytokines (TNF-alpha, IFN-gamma, Il-2, IL-12; Unanue, Curr. Opin.Immunol. 9:35-43, 1997; Mata and Paterson, J. Immunol. 163:1449-14456,1999) is induced, for which an antitumoral effectivity was detected. Forinstance, by the administration of L. monocytogenes, which weretransduced for the expression of tumor antigens, the growth ofexperimental tumors could antigen-specifically be inhibited (Pan et al.,Nat. Med. 1:471-477, 1995; Cancer Res. 59:5264-5269, 1999; Voest et al.,Natl. Cancer Inst. 87:581-586, 1995; Beatty and Paterson, J. Immunol.165:5502-5508, 2000). Virulence-attenuated Salmonella enterica strains,into which nucleotide sequences that encode tumor antigens have beenintroduced, could cause as tumor antigen-expressing bacterial carriersafter oral administration a specific protection against differentexperimental tumors (Medina et al., Eur. J. Immunol. 30:768-777, 2000;Zoller and Christ, J. Immunol. 166:3440-3450, 2001; Xiang et al., PNAS97:5492-5497, 2000). Recombinant Salmonella strains were also effectiveas prophylactic vaccines against virus infections (HPV) (Benyacoub etal., Infect. Immun. 67:3674-3679, 1999) and for the therapeutictreatment of a mouse tumor immortalized by a tumor virus (HPV) (Revaz etal., Virology 279:354-360, 2001). For the systemic tumor therapy,Salmonella strains were selected, which settle on specifically selectedtumor tissues (Murray et al., J. Bacteriol. 183:5554-5564, 2001). Intothese Salmonella strains as well as into Escherichia coli strains,nucleotide sequences that encode selected enzymes were introduced, andthese bacterial carriers were successfully used for GEDPT in vitro aswell as in vivo in experimental tumor systems (Pawelek et al., CancerRes. 57:4537-4544, 1997).

Inflammation tissues and in particular tumor tissues are characterizedby an increased angiogenesis in most cases chaotically proceeding in thetumor. In these newly formed vessels, soluble as well as particulatesubstances can be enriched, provided they have a low distribution volumeand thus a relatively long blood half-life. This enrichment (alsodesignated passive targeting) can be used for therapeutic methods(Sedlacek, Critical Reviews in Oncology/Hematology 37:169-215, 2001).

TECHNICAL OBJECT OF THE INVENTION

It is the object of the present invention to provide a pharmaceuticalcomposition, which has an increased effectiveness in the treatment ofproliferative diseases, in particular in the tumor therapy.

Basic Concept of the Invention and Findings the Invention is Based on.

For achieving the above technical object, the invention teaches anenveloped microorganism, in whose genome the following components areinserted and can be expressed: I) a nucleotide sequence that encodes adirectly or indirectly, antiproliferatively or cytotoxically activeexpression product or a plurality of said expression products; II) anucleotide sequence that encodes or is constitutively active for a bloodplasma protein under the control of an activation sequence that can beactivated in the microorganism; III) optionally, a nucleotide sequencethat encodes or is constitutively active for a cell-specific ligandunder the control of an activation sequence that can be activated in themicroorganism; IV) a nucleotide sequence for a transport system thatinduces expression of the expression products of components I) and II)and optionally III) on the outer surface of the microorganism or thatinduces secretion of the expression products of component I) andexpression of component II) and optionally component III) and that ispreferably constitutively active; V) optionally a nucleotide sequencefor a protein used for lysis of the microorganism in the cytosol ofmammalian cells and for the intracellular release of plasmids with atleast one or more components I) and VI) contained in the lysedmicroorganism; and VI) an activation sequence that can be activated inthe microorganism, and/or that is tissue-specific, tumor cell-specific,function-specific or not cell-specific, for expressing component I), anyof components I) to VI) being able to be present either single orseveral times, and either identical or different.

For the purpose of the invention, preferably enveloped microorganisms ascarriers for gene information and the use of said envelopedmicroorganisms for the prophylaxis and therapy of a proliferativedisease are described. The invention is based on the followingexperiences and technical developments.

Subject matter of the invention are therefore preferably envelopedmicroorganisms as carriers for nucleotide sequences for the treatment ofproliferative diseases, the following components having been insertedinto the microorganisms: I) at least one nucleotide sequence thatencodes at least one directly or indirectly, antiproliferatively orcytotoxically active expression product; II) at least one nucleotidesequence that encodes at least one blood plasma protein under thecontrol of at least one activation sequence that can be activated in themicroorganism; III) optionally, at least one nucleotide sequence thatencodes at least one cell-specific ligand under the control of at leastone activation sequence that can be activated in the microorganism; IV)at least one nucleotide sequence for at least one transport system thatmakes possible the expression of the expression products of componentsI) and II) and III) on the outer surface of the microorganism or thesecretion of component I), II) and III); V) optionally at least onenucleotide sequence for at least one protein used for lysis of themicroorganism in the cytosol of mammalian cells and for theintracellular release of plasmids contained in the lysed microorganism;and VI) at least one activation sequence what can be activated in themicroorganism or at least one tissue-specific, tumor cell-specific ornot cell-specific activation sequence, for expressing component I).

PREFERRED EMBODIMENTS OF THE INVENTION

Component I).

Component I) is at least one nucleotide sequence that encodes at leastone directly or indirectly, antiproliferatively or cytotoxically activeexpression product. Directly, antiproliferatively active expressionproducts in the meaning of the invention are for instance interferons,such as IFN-alpha, IFN-gamma, IFN-beta, interleukins, which inhibitimmune cells or tumor cells, such as IL-10, IL-12, proapoptotic peptidesor proteins, such as TNF-alpha, fas ligand, TNF-related apoptosisinducing ligand (TRAIL), antibodies or fragments of antibodies, whichact inhibitingly on or cytotoxically for an immune cell, a tumor cell ora cell of the tissue, from which the tumor originates, such asantibodies directed against i) a tumor-associated or tumor-specificantigen, ii) an antigen against lymphocytes, such as against the T cellreceptor, the B cell receptor, the receptor for the C40 ligand, the B7.1or B7.2, the receptor for an interleukin, such as IL-1, -2, -3, -4, -5,-6, -7, -8, -9, -10, -11, -12, -13, -14, -15 or -16, the receptor for aninterferon or the receptor for a chemokine, for instance for RANTES,MCAF, MIP-alpha, MIP-beta, IL-8, MGSA/Gro, NP-A-2 or IP-10, iii) atissue-specific antigen, such as against a tissue-specific antigen ofthe cells of mammary glands, kidneys, nevi, prostate, thyroid glands,tunica mucosa gastris, ovaries, cervix, vesica urinaria, anantiproliferatively active protein, such as the retinoblastoma protein(pRb=p110), or the related p107 and p130 proteins, orantiproliferatively active mutants of these proteins, the p53 proteinand analogous proteins or antiproliferatively active mutants of theseproteins, the p21 (WAF-1) protein, the p27 protein, the p16 protein, theGAAD45 protein, antiproliferatively active proteins of the Bcl2 family,such as bad or bak, cytotoxic proteins, such as perforin, granzyme,oncostatin, an antisense RNA or a ribozyme, specific for an mRNA, whichparticipates in the growth or the proliferation of a cell, for instancespecific for the mRNA that encodes a receptor, for a signal-transmittingenzyme, for a protein, which participates in the cell cycle, for atranscription factor or for a transport protein. Indirectly,proliferatively active proteins are for instance inductors of acuteinflammations and immune reactions, such as chemokines like RANTES(MCP-2), monocyte chemotactic and activating factor (MCAF), IL-8,macrophage inflammatory protein-1 (MIP-1-alpha, -beta), neutrophilactivating protein-2 (NAP-2), interleukins, such as IL-1, IL-2, IL-3,IL-4, IL-5, human leukemia inhibiting factor (LIF), IL-6, IL-7, IL-9,IL-11, IL-13, IL-14, IL-15, IL-16, cytokines, such as GM-CSF, G-CSF,M-CSF, enzymes for the activation or fission of the inactive pre-stageof a cytotoxic substance into a cytotoxic substance, said enzymes beingan oxidoreductase, a transferase, a hydrolase or a lyase. Examples forsuch enzymes are β-glucuronidase, β-galactosidase, glucose oxidase,alcohol dehydrogenase, lactoperoxidase, urokinase, tissueplasminogen-activator carboxy peptidase, cytosine deaminase,deoxycytidine kinase, thymidine kinase, lipase, acidic phosphatase,alkaline phosphatase, kinase, purine nucleoside phosphorylase, glucoseoxidase, lactoperoxidase, lactate oxidase, penicillin V amidase,penicillin G amidase, lisozyme, β-lactamase, aminopeptidase,carboxypeptidase A, B or G2, nitroreductase, cytochrome P450 oxidase.According to the invention the enzyme can originate from a virus, abacterium, a yeast, a mollusk, an insect or a mammal. Preferably suchenzymes are used, which originate from man. Furthermore, such nucleicacid constructs are preferred in the meaning of the invention, whichencode a fusion product of a cell-specific ligand with an enzyme, and/orproteins, which inhibit angiogenesis, for instance plasminogen activatorinhibitor-1 (PAI-1), PAI-2 or PAI-3, angiostatin or endostatin,interferon-alpha, -beta or -gamma, interleukin 12, platelet factor 4,thrombospondin-1 or -2, TGF-beta, TNF-alpha, vascular endothelial cellgrowth inhibitor (VEGI). In the meaning of the invention, the componentI) may represent one or more nucleotide sequences that encode one ormore identical or different, directly or indirectly, proliferatively orcytotoxically active proteins. Preferred are combinations of proteins,which have an additive or synergistic effect. Additive or synergisticeffects can for instance be expected for the following combinations ofdifferently active proteins: cytotoxic proteins and proapoptoticproteins, enzymes and cytotoxic and/or proapoptotic proteins,antiangiogenetic proteins and cytotoxic and/or proapoptotic proteins,inductors of inflammations and enzymes or cytotoxic, proapoptotic and/orantiangiogenetic proteins.

Component II).

Component II) is a nucleotide sequence that encodes at least one bloodplasma protein under the control of an activation sequence that can beactivated in the microorganism. Preferred are human blood plasmaproteins, namely those, which have an average dwell time in the blood ofmore than 24 hours. To these belong in particular for instance albumin(nucleotide 1-2258; Hinchliffe et al., EP 0248637-A, Sep. 12, 1987),transferrin (nucleotide 1-2346; Uzan et al., Biochem. Biophys. Res.Commun. 119:273-281, 1984; Yang et al., PNAS-USA 81:2752-2756, 1984),ceruloplasmin (Baranov et al., Chromosoma 96:60-66, 1987), haptoglobin(nucleotide 1-1412; Raugei et al., Nucleic Acids Res. 11:5811-5819,1983; Yang et al., PNAS-USA 80:5875-5879, 1983; Brune et al., NucleicAcids Res. 12:4531-4538, 1984), hemoglobin alpha (nucleotide 1-576;Marotta et al., PNAS-USA 71:2300-2304, 1974; Chang et al., PNAS-USA74:5145-5149, 1977), hemoglobin beta (nucleotide 1-626; Marotta et al.,Prog. Nucleic Acid Res. Mol. Biol. 19:165-175, 1976; Marotta et al., J.Biol. Chem. 252:5019-5031, 1977), alpha2-macroglobulin (nucleotide1-4599; WO 9103557 A, 21/3/1991). Thereto belong, however, other bloodplasma proteins, too, such as alpha-1-lipoprotein, alpha-2-lipoprotein,beta-1-lipoprotein. The expression of at least one of these plasmaproteins by the microorganism according to the invention has as aconsequence that the microorganism is received after systemicadministration—in particular after injection into the blood circulationsystem—to a lower degree by phagocytosing cells, thus can stay longer inthe blood and can be enriched in the tumor vessel system or in thevessels of a chronic inflammation.

Component III).

Component III) is a nucleotide sequence that encodes a cell-specificligand under the control of an activation sequence that can be activatedin the microorganism. The specificity of this ligand depends on the kindof the proliferative disease, for which the microorganism is used, andon the cells or the tissue, with which component I) is to be broughtinto contact in the microorganism, in order to achieve the therapeuticeffectivity. For instance, in tumor diseases, ligands with specificityfor tumor cells are used, i.e. for tumor-associated or tumor-specificantigens or tumor endothelium cells or for tissue cells, from which therespective tumor originates, for instance for cells of the thyroidgland, the prostate, the ovary, the mammary, the kidney, the tunicamucosa gastris, the nevi, the cervix, the vesica urinaria; for chronicinflammations, cellular autoimmune diseases and rejections oftransplanted organs, ligands either with specificity for macrophages,dendritic cells, T lymphocytes or for activated endothelium cells. Suchligands are for instance specific antibodies or antigen-bindingfragments of these antibodies, growth factors, interleukins, cytokinesor cell adhesion molecules selectively binding to tumor cells, toleukemia cells, to tumor endothelium cells, to tissue cells, tomacrophages, dendritic cells, T lymphocytes or to activated endotheliumcells.

Component IV).

Component IV) is a nucleotide sequence that encodes a transport system,which permits the expression of the expression products of componentsI), II) and/or III) on the outer surface of the microorganism. Therespective component can as an option either be secreted or expressed onthe membrane of the microorganism, i.e. membrane-bound. Components II)and III) are preferably expressed membrane-bound. Such transport systemsare for instance the hemolysin transport signal of E. coli (nucleotidesequence containing hlyA, hlyB and hlyD under the control of thehly-specific promoter, Gentschev et al., Gene 179:133-140, 1996). Thefollowing transport signals can be used: for the secretion, theC-terminal hlyA transport signal, in presence of hlyB and hlyD proteins;for the membrane-bound expression, the C-terminal hlyA transport signal,in presence of the hlyB protein; the hemolysin transport signal of E.coli (nucleotide sequences containing hlyA, hlyB and hlyD under thecontrol of a not hly-specific bacterial promoter); the transport signalfor the S-layer protein (Rsa A) of Caulobacter crescentus; for thesecretion and for the membrane-bound expression, the C-terminal RsaAtransport signal (Umelo-Njaka et al., Vaccine 19:1406-1415, 2001); thetransport signal for the TolC protein of Escherichia coli (the TolCprotein was described by Koronakis et al., Nature 405:914-919, 2000) andby Gentschev et al., Trends in Microbiology 10:39-45, 2002)); for themembrane-bound expression, the N-terminal transport signal.

Component V).

Component V) is a nucleotide sequence that encodes at least one lyticfor a protein, which is expressed in the cytosol of a mammalian cell andlyses the microorganism for the release of the plasmids in the cytosolof the host cell. Such lytic proteins (endolysins) are for instanceListeria-specific lysis proteins, such as PLY551 (Loessner et al., Mol.Microbiol. 16:1231-41, 1995), the Listeria-specific holin under thecontrol of a listerial promoter. A preferred embodiment of thisinvention is the combination of different components V), for instancethe combination of a lysis protein with a holin.

Component VI).

Component VI) represents an arbitrary activator sequence, which controlsthe expression of component I). For the expression of component I) onthe outer surface of the microorganism, component VI) is one ofactivations sequences that can be activated in the bacterium and that isknown to the man skilled in the art. Such activation sequences are forinstance constitutively active promoter regions, such as the promoterregion with ribosomal binding site (RBS) of the beta-lactamase gene ofE. coli or of the tetA gene (Busby and Ebright, Cell 79:743-746, 1994),promoters that can be induced, preferably promoters that become activeafter reception in the cell. To the latter belongs the actA promoter ofS. monocytogenes (Dietrich et al., Nat. Biotechnol. 16:181-185, 1998) orthe pagc promoter of L. monocytogenes (Bumann, Infect. Immun.69:7493-7500, 2001). Preferred are activator sequences, which, afterrelease of the plasmids of the bacterial carrier in the cytosol of thetarget cell, are activated in this cell. For instance, the CMV enhancer,the CMV promoter, the SV40 promoter or any other promoter or enhancersequence known to the man skilled in the art can be used. Preferred arefurther cell-specific or function-specific activator sequences. Theselection of the cell-specific or function-specific activator sequencedepends on the cell or the tissue, wherein the bacterial carrier or theplasmids released from the bacterial carrier are to express componentI). Such activator sequences are for instance tumor cell-associatedactivator sequences (thereto belong activator sequences of the genes formidkine, GRP, TCF-4, MUC-1, TERT, MYC-MAX, surfactant protein,alpha-fetoprotein, CEA, tyrosinase, fibrillary acidic protein, EGR-1,GFAP, E2F1, basic myelin, alpha-lactalbumin, osteocalcin, thyroglobulinand PSA (McCormick, Nature Reviews Cancer 1:130-141, 2001), endotheliumcell-specific activator sequences of the genes for proteins, which arepreferably expressed by endothelium cells (Sedlacek, Critical Reviews inOncology/Hematology 37:169-215, 2001), such as VEGF, von Willebrandfactor, brain-specific endothelial glucose-1 transporter, endoglin, VEGFreceptors, in particular VEGF-R1, VEGF-R2, and VEGF-R3, TIE-2, PDECGFreceptors, B61, endothelin-1, endothelin-B, mannose 6-phosphatereceptors, VCAM-1 and PE-CAM-1, activator sequences of the genes forproteins, which are preferably expressed in such tissue cells from whichthe tumor cells of a patient originate (thereto belong proteinsexpressed in cells of the breast tissue (for instance MUC-1,alpha-lactalbumin), the thyroid gland (for instance thyroglobulin), theprostate (for instance kallikrein-2, androgen receptors, PSA), theovary, the nevi (for instance tyrosinase), and the kidney, activatorsequences of the genes for proteins, which are expressed in macrophages,dendritic cells or lymphocytes, such as interleukins, cytokines,chemokines, adhesion molecules, interferons, receptors for interleukins,cytokines, chemokines, or interferons, activator sequences, which areactivated by hypoxia, such as the activator sequence for VEGF or forerythropoietin.

The insertion of components I) to VI) into the microorganisms is made bymolecular biological methods known to the man skilled in the art. Forinstance, for the use of bacteria as carriers, the man skilled in theart is familiar with how the components are inserted into suitableplasmids, and how these plasmids are introduced into the bacteria.

According to the present invention, these microorganisms areadministered to a patient for the prophylaxis or therapy of aproliferative disease, such as a tumor, a leukemia, a chronicinflammation, an autoimmune disease or the rejection of an organtransplant. For treating such a disease, the microorganisms according tothe invention are administered in a suitable preparation locally orsystemically, for instance into the blood circulation, into a bodycavity, into an organ, into a joint or into the connective tissue. Inorder, with systemic administration, in particular with administrationinto the blood circulation, to reduce the undesired reception of themicroorganisms by the so-called reticuloendothelial system beyond theeffect of component II) and to extend the blood dwell time of themicroorganisms, the microorganisms can be suspended in a solution ofsubstances, which have a long blood dwell time. To the suspensionfollows an incubation. The suspension and incubation of themicroorganisms can for instance take place in blood plasma or bloodserum. The suspension and incubation is however preferably performed insolutions of substances or solutions of mixtures of substances, whichhave a long blood dwell time. To these substances belong for instancealbumin, transferrin, prealbumin, hemoglobin, haptoglobin,alpha-1-lipoprotein, alpha-2-lipoprotein, beta-1-lipoprotein,alpha-2-macroglobulin, polyethylene glycol (PEG), PEG conjugates withnatural or synthetic polymers, such as polyethylene imine, dextran,polygeline, hydroxyethyl starch.

By the suspension and incubation in such a solution, an adsorption ofthe substances to the surface of the microorganisms according to theinvention takes place. A coating of the microorganisms with thesesubstances can however also be achieved by conjugation. The methods ofthe conjugation are summarized in Sedlacek et al., Contributions toOncology 32:1-132, 1988.

The coating by adsorption takes place for instance by suspension of themicroorganisms in a solution preferably containing 0.1 to 50% of thecoating substances over a period of time of preferably 10 minutes to 24hours and a temperature of preferably 4 degrees Celsius.

According to the invention, as microorganisms, preferably bacteria areused, the virulence of which has been reduced. Further preferred arebacteria selected from a group containing Escherichia coli, Salmonellaenterica, Yersinia enterocolitica, Vibrio cholerae, Listeriamonocytogenes, Shigella.

Microorganisms in conjunction with the invention are further membraneenvelopes, so-called ghosts, of live or existing microorganisms. Suchmembrane envelopes are for instance produced according to EPA 0540525.

Subject matter of the invention are medicament preparations containingthe microorganisms according to the invention and the use of thismedicament for the prophylaxis and/or therapy of a proliferativedisease. A proliferative disease in the meaning of the present inventionis a disease with an escalating or uncontrolled cell proliferation, forinstance a tumor disease such as a carcinoma or a sarcoma, a leukemia, achronic inflammation, an autoimmune disease or the rejection of an organtransplant. For the prophylaxis or therapy of a disease, themicroorganisms according to the invention are locally or systemicallyadministered to a patient in the medicament preparation in a dose ofpreferably 100 germs to 100 million germs.

The term enveloped means that on the outside of the membrane of themicroorganism, a multitude of identical or different molecules(expressed and/or selected according to one or more of features I) toIII)), as described above, can be provided, the geometric coverage ratebeing between 0.001 and 1, in particular between 0.01 and 1, forinstance between 0.1 and 1. The geometric coverage rate can becalculated from the ratio of the total area of all molecules, in aradial (related to a center of the microorganism) projection into thesurface of the microorganism, divided by the surface area of themicroorganism. Usually, as a simplification, a spherical surface of themicroorganism is assumed, and the calculation is based on the volume ofthe microorganism. The feature “enveloped” is facultative, ifapplicable.

EXAMPLES OF EXECUTION Example 1 Construction of a Bacteria Strain forthe Membrane-Bound Expression of Human Albumin and Beta-glucuronidase

In this example, the way to the bacteria strain St21-bglu is described.This attenuated Salmonella typhi Ty21a strain (carrier approved forhuman use) expresses by means of the hly secretion machinery of E. colimembrane-bound fusion proteins of human beta-glucuronidase and hlyA andhuman albumin and hlyA. The construction is based on the alreadypublished plasmids pMOhlyl (Gentschev et al., Behring Inst. Mitt. 57-66,1994) and pGP704 (Miller and Mekalanos, J. Bacteriol. 170:2575-2583,1988). The strain permits by passive targeting (Bermudes et al., Adv.Exp. Med. Biol. 465:57-63, 2000) an enrichment of beta-glucuronidase atthe tumor and thus a fission restricted to the tumor tissue of prodrugsto be activated by beta-glucuronidase.

A membrane-bound expression can take place in salmonellae by fusion ofthe protein to the C-terminus of the hlyA secretion protein in presenceof the hlyB protein, however in absence of a completely functional hlyDprotein. However, the hlyD must not completely be missing, sinceotherwise there will not be generated a connection between the secretionmachinery and the TolC protein of the outer membrane (Spreng et al.,Mol. Microbiol. 31:1596-1598, 1999). In these examples one of thepossible modifications of the hlyD protein for the membrane-boundexpression is indicated. First the vector pMOhly DD is constructed,wherein no functional hlyD protein is produced. For this purpose, partof the hlyD gene is removed from the vector pMOhlyl by the endonucleasesDraIII and ApaI. After the restriction digestion, the ends are digestedby 3′-5′ exonuclease, and the 10,923 bps fragment is religated.Subsequently the beta-glucuronidase gene is cloned into this vectorin-frame to the hlyA gene. For this purpose, the cDNA of bglu (GenBankAccession (Gb): M15182) from a cDNA bank was amplified with thefollowing primers by polymerase chain reaction (PCR): bglu 5′: ATGCATTGCAGGGCGGGATGCTGTACC bglu 3′: ATGCAT AAGTAAACGGGCTGTTTTCCAAAC

The regions being complementary to the cDNA of beta-glucuronidase areunderlined, the information for the generated NsiI position is initalics (this kind of representation will also be used in the following;the oligonucleotide sequences are shown here, as in the following, as5′-3′). The primers are selected such that the gene is amplified withoutthe signal sequence. The product (1,899 bps) is subcloned with asuitable PCR cloning kit, and then the ≈1,890 bps fragment is extractedvia NsiI digestion. Subsequently, the NsiI fragment is cloned into theNsiI-cut vector pMOhly DD. This results in the vector pMO DDbglu (FIG.1). (When the NsiI fragment is cloned into the NsiI-cut vector pMOhlyl,the plasmid pMO bglu is obtained permitting a secernation of the fusionprotein). In the second part the integration vector for the chromosomalintegration of the albumin hlyA fusion is produced. In a first step, thevector pMOhly alb is produced. This vector being based on pMOhlylcarries a fusion of the albumin cDNA with the hlyA gene. For cloning,the cDNA of the albumin gene (Gb: A06977) from a commercially availablecDNA bank is amplified by means of PCR and the following primersgenerating NsiI: 5′: ATGCAT GGGTAACCTTTATTTCCCTTC 3′: ATGCATAGCCTAAGGCAGCTTGACTTG-

The 1,830 bps fragment is subcloned and then cut with NsiI. The 1,824bps fragment is now ligated in NsiI-digested pMOhlyl. The completedplasmid pMOhly alb thus expresses hlyB, hlyD and a fusion protein fromalbumin and hlyA. For experiments regarding the dwell time, the NsiIfragment can alternatively also be inserted into the vector pMO DD, thisvector has the name pMO DDalb. In the further course, a modification ofthe already described cloning strategy is used for the integration inthe salmonella chromosome (Miller et Mekalanos, J. Bacteriol.170:2575-2583, 1988). For this purpose, first the aroA gene ofsalmonella was cloned into the vector pUC18 (PCR with the followingprimers: primer 5′: ATGGAATCCCTGACGTTACAACCC, primer 3′:GGCAGGCGTACTCATTCGCGC

-   -   blunt cloning of the 1,281 bps fragment into the HincII        interface of pUC18). Subsequently, a 341 bps fragment located in        aroA was removed by HincII digestion and subsequent religation.        This vector was called pUC18 aroA′. Then the alb-hlyA fusion        gene was cloned together with the promoter sequence located on        pMOhly into the vector pUC18aroA′. For this purpose, the vector        pMOhly alb is digested with AacII and SwaI and then treated with        a 3′-5′ exonuclease. The 3,506 bps blunt fragment is extracted        and ligated in HincII-digested pUC18aroA′. This produces the        vector pUCaro-alb. Now, the alb-hlyA fragment flanked by aroA is        cloned with all the activator sequences from the vector        pUCaro-alb into the vector pGP704. For this purpose, pUCaro-alb        is digested with HindIII and then treated with 5′-3′ exonuclease        (blunt). Subsequently, EcoRI digestion is performed, and the        4,497 fragment is ligated into the EcoRI/EcoRV (blunt) digested        vector pGP704 (EcoRI/RV fragment: 6,387 bps). The integration        vector pGParo-alb (FIG. 2) is obtained. The vector is        transformed into the E. coli strain SM101pir. This strain        permits the vector to replicate, since it forms the P protein        necessary for replication. The vector is now transferred via        conjugation into the acceptor strain Salmonella typhi Ty21a not        permitting a replication of the vector. Therefore, by        tetracycline selection, only those bacteria are selected that        have integrated the vector chromosomally. The verification of        the cytoplasmic albumin production takes place by Western blot        analysis of the bacterium lysate. This strain St21-alb expresses        the alb-hlyA fusion, but can neither secern nor express it on        the membrane in this form. For this purpose, for the        membrane-bound expression, in addition a plasmid with functional        hlyB (as pMO DDbglu) or functional hlyB and hlyD (as pMO bglu)        needs to be present.

In this example, the plasmid pMO DDbglu with the strain St21-alb isused. This results in the strain St21-alb pMO DDbglu expressing by meansof the hly secretion system human albumin as well as humanbeta-glucuronidase on the membrane. This strain can then be used for theprodrug conversion in the meaning of the patent.

Example 2 Construction of a Bacteria Strain Enveloped with Albumin-hlyAFusion for Supplying the Genetic Information of HumanBeta-glucuronidase.

The bacteria strain described in this example is intended to supply bymeans of the passive targeting DNA that encodes human beta-glucuronidasefor tumor cells, which are then to be expressed in the tumor cells. Inorder to obtain a strain being particularly easy to handle, in thisexample a slightly modified strain as in Example 1 is used for themembrane expression of albumin. The gene that encodes albumin-hlyA aswell as the information for hlyB is to be chromosomally integrated.Thereby, this strain expresses constitutively membrane-bound albumin.

For this purpose, the vector pMOhly alb described above is digested byBsrBI and EcoRI and then treated with 5′-3′ exonuclease. This digestionproduces a 5,815 bps fragment with blunt ends containing the completeprokaryontic activation sequence and the genes hlyC, alb-hlyA and hlyB,not however hlyD. This fragment can now bluntly be inserted into theHincII interface of the vector pUC18aroA∝0 described above. Thereby thevector pUCaro-alb-B is obtained. By an EcoRI-NruI digestion, the 6,548bps fragment can again be inserted into the EcoRI-EcoRV-digested vectorpGP704 (FIG. 3). The further procedure (replication and integration inS. typhi Ty21a) corresponds to the above strategy. The resulting strainSt21-alb-B expresses constitutively membrane-bound albumin-hlyA fusionprotein. If a vector that encodes hlyD is transfected, the albumin-hlyAfusion protein is secerned. The plasmid for supplying the DNA thatencodes beta-glucuronidase is based on the commercially available vectorpCMVbeta (Clontech). For the construction, first a fusion of the bglugene with a secretion signal must be used. In this example, the signalpeptide of the tPA precursor molecule is to be used. This signal peptidepermits a particularly efficient production and secretion of fusionproteins. For cloning the fusion, in a first step the 5′ UTR of the tPAcDNA (Gb E02027) is amplified up to the end of the region that encodesthe signal peptide with the following primers via PCR (amplificationwith blunt generating polymerase): 5′: GCGGCCGC AGGGAAGGAGCAAGCCGTGAATTT3′: AGCTT AGATCTGGCTCCTCTTCTGAATC

The generated 166 bps fragment is ligated into the HindIII-digested,51-3′ exonuclease-treated commercially available vector pcDNA3(Invitrogen). The ligation is made in the forward orientation. Thereby,the region that encodes tPA signal sequence can completely be cut outvia a NotI digestion from the generated plasmid pCDNAtp. This 237 bpsfragment is now ligated with the 3,760 bps fragment of the vectorpCMVbeta after NotI digestion (contains vector backbone). The generatedplasmid pCMVtp (3,972 bps) can now be used for the expression ofheterologous fusion proteins. For the generation of the plasmid pCMVbglu, a bps fragment of the bglu (Gb M15182) gene (without sequence forsignal peptide) from a suitable cDNA bank is amplified with thefollowing primers generating SpeI: 5′: ACTAGT CAGGGCGGGATGCTGTACCCCCAG3′: ACTAGT CTTGCTCAAGTAAACGGGCTGTTTTC.

After SpeI-digestion, the 1,899 bps fragment is ligated into theSpeI-digested vector pCMVtp. The generated plasmid pCMVtp bglu encodesnow an N-terminal fusion of the tPA signal peptide with the region ofthe mature protein of beta-glucuronidase. After determination of thecorrect position, the plasmid pCMVtp bglu (FIG. 4) is transformed intothe strain St21-alb-B. This strain permits now a supply of the DNA tothe tumor tissue by means of passive targeting, and the expression ofthe DNA by transfected tumor cells permits then a conversion of suitableprodrugs.

Example 3 Construction of a Strain Enveloped with Albumin-TolC Fusionwith Membrane-Bound Expression of the Extra-Cellular Domain of fas andSupply of an Enzyme Converting Prodrug

The strain shown in this example unites the features shown in Example 2with a specific targeting at (tumor) cells expressing fas ligand (fasL).It is possible, with this strain, to specifically attack fasL-expressingtumor cells, such as in certain breast tumors (Herrnring et al.,Histochem. Cell. Biol. 113:189-194, 2000). fasL expression by tumorcells was postulated as a potential mechanism for immune escape, sincethese cells can eliminate actively attacking, fas-expressing lymphocytes(Muschen et al., J. Mol. Med. 78:312-325, 2000). With the strain shownhere, these tumor cells being very problematic for a therapy canspecifically be attacked and then eliminated by an apoptosis-independentmechanism. The carrier strain is based in this example on a fusion ofalbumin with the TolC protein of E. coli. Thereby, a membrane-boundexpression of albumin is achieved. The membrane-bound expression of theextracellular domain of fas takes place via the plasmid pMOhlyDD, andfor the supply the plasmid pCMV-bglu described above is used. The firststep comprises the generation of the carrier strain expressing TolCalbumin. First the gene for the fusion protein is generated, and thenthis gene is integrated, according to the above examples, via successivecloning in pUCaroA′ and pGP704 into the salmonella genome. The TolC genefor E. coli, including the natural promoter, is present in the plasmidpBRtolC. This was amplified by means of the following primers generatingSalI from the vector pAX629 (contains tolC gene, region in the vectorcorresponds to Gb X54049 pos. 18-1914): 5′tol: TAACGCCCTAT GTCGACTAACGCCAACCTT, 3′tol: AGAGGAT GTCGAC TCGAAATTGAAGCGAGA.

The 1,701 bps fragment was inversely ligated after fission with SalIinto the SalI interface of the vector pBR322 (Gb J01749), thus the tetgene being interrupted. Due to the known crystal structure of TolC(Koronakis et al., Nature 405:914-919, 2000), the insertion ofheterologous DNA into the singular KpnI interface in the tolC genepermits the expression of the encoded heterologous fusion protein in anextracellular loop on the outer membrane. For the expression of albumin,the albumin gene is amplified from the cDNA (Gb A06977) by means of thefollowing primers generating KpnI: 5′: GGTACC CGAGATGCACACAAGAGTGAGG 3′:GGTACC TAAGCCTAAGGCAGCTTGACTTGC.

After KpnI digestion of the 1,770 bps fragment, the DNA can be insertedinto the KpnI-cut vector pBRtolC. The reverse orientation (in frame totolC) results then in the vector pBRtolC-alb. The gene for thetolC-albumin fusion is ligated now in reversed orientation via EcoRV andPshAI (fragment 3,970 bps) into the HincII interface of the vectorpUCaroA′. The obtained vector pUCaro-alb-tol (7,596 bps) is nowlinearized with HindIII, treated with 5′-3′ exonuclease and thendigested with EcoRI. The 4,961 bps fragment is then inserted into theEcoRI-EcoRV-digested vector pGP704 (FIG. 5). After conjugation(according to Example 1) the strain St21-tol-alb is obtained. Now theplasmid is used for the membrane-bound expression of a fas (CD95)-hlyAfusion protein by means of the hlyB component of the E. coli type Isecretion machinery. For this purpose, first the section that encodesthe extracellular region of the fas gene (Gb: M67454) is amplified withthe following primers generating NsiI: 5′: ATGCAT TATCGTCCAAAAGTGTTAATGC3′: ATGCAT TAGATCTGGATCCTTCCTCTTTGC.

The 477 bps fragment is digested with NsiI and inserted into theNsiI-digested vector pMOhly DD in frame to the hlyA gene. The obtainedvector pMO DD-fas (FIG. 6) thus produces after transformation into asalmonella strain a membrane-bound fas fragment, which with suitablefolding can bind to fasL-expressing cells. Thus, these salmonellae canbe enriched at fasL-expressing cells, such as tumor cells.

For killing the fasL tumor cells, now the plasmid pCMV bglu (Example 2)is also transfected into the salmonellae. Thereby, as in the aboveexample, after expression of the beta-glucuronidase by tumor cells, aprodrug-drug-mediating tumor therapy is possible. The bettereffectiveness of this example compared to the previous example dependsin a decisive way on the correct folding of the extracellular domain offas. In lieu of fas, fasL-specific fab fragments of monoclonalantibodies (which can correctly be folded in bacteria) can be used inthe same approach as described here. This example shows that by means ofthis technique, the construction of strains with nearly any cellspecificity is possible via the use of suitable specific fab fragments.

LEGEND OF THE FIGURES

FIG. 1: vector pMO Dbglu

FIG. 2: vector pGParoalb

FIG. 3: pGParo-alb-B

FIG. 4: pCMVtp bglu

FIG. 5: pGParo-alb-tol

FIG. 6: pMO DD-fas

1. A microorganism in whose genome the following components are insertedand can be expressed: a) a nucleotide sequence that encodes a direct orindirect antiproliferative or cytotoxically active expression product ora plurality of said expression products, b) a nucleotide sequence thatencodes for a blood plasma protein under the control of an activationsequence that can be activated in the microorganism, or that isconstitutively active, c) a nucleotide sequence that encodes for acell-specific ligand under the control of an activation sequence thatcan be activated in the microorganism, or is constitutively active, d) anucleotide sequence for a transport system that induces expression ofthe expression products of components a) and b) and optionally c) on theouter surface of the microorganism or that induces secretion of theexpression products of component a) and expression of compenent b) andoptionally c) and that is preferably constitutively active, e) anucleotide sequence for a protein used for lysis of the microorganism inthe cytosol of mammalian cells and for the intracellular release ofplasmids with at least one or more components a) and f) contained in thelysed microorganism, and f) an activation sequence that can be activatedin the microorganism, or that is tissue cell-specific, tumorcell-specific, function-specific or not cell-specific, for expressingcomponent a), wherein any of components a) to f) is present either onceor several times, and are either identical or different.
 2. Themicroorganism according to claim 1, wherein the microorganism is avirus, a bacterium or a monocellular parasite.
 3. The microorganismaccording to claim 1 or 2, wherein the virulence of the microorganism isreduced.
 4. The microorganism according to claim 1, wherein themicroorganism is a gram-positive or gram-negative bacterium.
 5. Themicroorganism according to claim 1, selected among a group consisting ofEscherichia coli, Salmonella, Yersinia enterocolitica, Vibrio cholerae,Listeria monocytogenes, and Shigella.
 6. The microorganism according toclaim 1, wherein the microorganism is the envelope of a bacterium. 7.The microorganism according to claim 1, wherein component a) encodes atleast one protein selected from the group consisting of interferons;interleukins; proapoptotic proteins; antibodies and antibody fragments,which act inhibitingly on or cytotoxically for an immune cell, a tumorcell or for cells of the tissue, from which the tumor originates;antiproliferatively active proteins; cytotoxic proteins; inductors of aninflammation, in particular interleukins, cytokines or chemokines;viral, bacterial enzymes or enzymes that originate from a yeast, amollusk, a mammal or man for the activation or fission of an inactivepre-stage of a cytostatic substance into the cytostatic substance;fusion products from a cell-specific ligand and an enzyme; andinhibitors of the angiogenesis.
 8. The microorganism according to claim1, wherein component b) encodes at least one blood plasma proteinselected from a group consisting of albumin, transferrin, haptoglobin,hemoglobin, alpha-1-lipoprotein, alpha-2-lipoprotein, beta-1-lipoproteinand alpha-2-macroglobulin.
 9. The microorganism according to claim 1,wherein component c) encodes at least one ligand specific for a targetorganism selected from a group consisting of tumor cells; tumorendothelium cells; tissue cells, from which originates a tumor;activated endothelium cells; macrophages; dendritic cells; andlymphocytes.
 10. The microorganism according to claim 1, whereincomponent c) encodes at least one ligand specific for a tissue cell typeof tissues selected from a group consisting of thyroid gland, mammary,salivary gland, lymph gland, mammary, tunica mucosa gastris, kidney,ovary, prostate, cervix, vesica urinaria, and nevus.
 11. Themicroorganism according to claim 1, wherein component d) encodes thehemolysin transport signal of Escherichia coli, the S-layer (Rsa A)protein of Caulobacter crescendus, or the ToiC protein of Escherichiacoli.
 12. The microorganism according to claim 1 wherein component e)encodes a lytic protein of gram-positive bacteria, lytic proteins ofListeria monocytogenes, PLY551 of Listeria monocytogenes or holin ofListeria monocytogenes.
 13. The microorganism according to claim 1,wherein at least one substance is bound to the microorganism which has along blood dwell time and which is selected among the group consistingof albumin, transferrin, prealbumin, hemoglobin, haptoglobin,alpha-1-lipoprotein, alpha-2-lipoprotein, beta-1-lipoprotein,alpha-2-macroglobulin, polyethylene glycol (PEG), PEG conjugates withnatural or synthetic polymers, such as polyethylene imine, dextran,polygeline, hydroxyethyl starch and mixtures of these substances,wherein the binding of the substance or substances takes place byphysisorption, chemisorption or covalently.
 14. A plasmid or expressionvector comprising components a) b) d) and f) and one or more ofcomponents c) and e).
 15. A method for the production of an organismaccording to claim 1, wherein a plasmid or expression vector comprisingcomponents a) b) d) and f) and one or more of components c) and e, isproduced, and a microorganism is transformed with this plasmid.
 16. Apharmaceutical composition comprising a microorganism according toclaim
 1. 17. A pharmaceutical composition for the prophylaxis and/ortherapy of a disease, which is caused by an uncontrolled cell divisioncomprising a tumor disease comprising a prostate carcinoma, an ovarycarcinoma, a mamma carcinoma, a stomach carcinoma, a kidney tumor, athyroid gland tumor, a melanoma, a cervix tumor, a bladder tumor, asalivary gland tumor or a lymph gland tumor, a leukemia, aninflammation, an organ rejection, or an autoimmune disease, wherein thecomposition comprises the microorganism according to claim
 1. 18. Thecomposition of claim 17 wherein the composition is used for removal of atumor as well as of healthy tissue, from which originates the tumor. 19.A method for the production of a pharmaceutical composition according toclaim 16, wherein an enveloped microorganism according to claim 1 isprepared in a physiologically effective dose with one or morephysiologically tolerated carrier substances for oral, intramuscular,intraveneous or intraperitoneal administration.